CN114712264B

CN114712264B - Synergistic stable sodium hyaluronate composite solution composition and preparation thereof

Info

Publication number: CN114712264B
Application number: CN202210637888.4A
Authority: CN
Inventors: 徐林; 金玉; 徐念沁; 居丽娜; 徐松泉; 曹晶晶
Original assignee: Nanjing Tzong Blotechnology Co ltd
Current assignee: Nanjing Tzong Blotechnology Co ltd
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-09-09
Anticipated expiration: 2042-06-08
Also published as: CN114712264A

Abstract

The invention discloses a synergistic stable sodium hyaluronate composite solution composition and a preparation thereof, wherein the sodium hyaluronate composite solution composition comprises non-crosslinked sodium hyaluronate, a hyaluronidase inhibitor, a solution stabilizer, an antioxidant and a pH regulator; wherein the hyaluronidase inhibitor is an amino acid. According to the invention, amino acid is used as a hyaluronidase inhibitor for the first time and is used as an auxiliary material to be combined with non-crosslinked sodium hyaluronate, so that the problems of fast in-vivo hydrolysis and short half-life period of sodium hyaluronate are solved; the purpose of prolonging the retention time of the non-crosslinked sodium hyaluronate in vivo is achieved, and the adopted amino acid is used as the hyaluronidase inhibitor, so that the hyaluronidase inhibitor has good performance of inhibiting the activity of hyaluronidase, is safe and reliable, can effectively promote the generation of collagen while inhibiting the activity of the hyaluronidase, and has good medical and cosmetic values and economic values.

Description

Synergistic stable sodium hyaluronate composite solution composition and preparation thereof

Technical Field

The invention belongs to the technical field of biology, relates to sodium hyaluronate and a hydrolase inhibitor thereof, and particularly relates to a synergistic and stable sodium hyaluronate composite solution composition containing a hyaluronidase inhibitor and a preparation thereof.

Background

The injection uses special instrument 'water light gun' to inject the needed injection into the corium layer closely under the epidermis, and after injection, the skin can become moist and bright. The injection can improve skin problems such as canthus wrinkle, mouth corner wrinkle, and neck line caused by skin dryness and skin elasticity decrease, and can moisten skin and enrich water. The main purpose of the water light injection is to supplement hyaluronic acid, which is a natural substance constituting the human body and has no side effect and rejection reaction to the human body. With the increase of age, the content of hyaluronic acid in a human body can be lost, and research data shows that compared with skin of 20-30 years old, the content of hyaluronic acid in the human body is 75% at the age of 30-40 years old, and is only 60% above 40 years old. The loss of hyaluronic acid can cause skin aging, skin elasticity reduction and fine lines generation, and the injection of hyaluronic acid can improve the water retention capacity, so that the skin is moistened and bright, and the hyaluronic acid skin moisturizing cream plays an important role in skin moisturizing.

After entering the dermis, the hyaluronic acid injected by water light hydrates with cells to promote blood microcirculation and the absorption of nutrients by the skin, but the hyaluronic acid itself is continuously diluted and absorbed, so that the time for which the water light injection can be maintained is limited. Generally, the time for maintaining after the first injection is short, about 1-2 months, and the effect can be maintained for about 1 year after three consecutive water light injections; however, the maintenance time of the skin care product varies according to the individual constitution, skin quality and living habits, and the maintenance time of the effect of the water-light direct injection is short, about 3 months or so for people with dry skin and being carefree.

Nowadays, the main ingredient of the water-luster product in the market is sodium hyaluronate with the chemical formula of (C) ₁₄ H ₂₀ NO ₁₁ Na) n, the chemical structure is shown in figure 1. Sodium hyaluronate is an inherent component in human body, is glucan aldehydic acid and has no species specificity; it is widely present in tissues such as placenta, amniotic fluid, crystalline lens, articular cartilage, dermal layer of skin, etc., and is distributed in cytoplasm and intercellular substance, and has lubricating and nourishing effects on cells and cell organs contained therein. The sodium hyaluronate belongs to a high molecular polymer and has strong lubricating feeling and film forming property, so that the skin care product containing the sodium hyaluronate has obvious lubricating feeling and good hand feeling when being smeared. The macromolecular sodium hyaluronate forms a layer of breathable film on the surface of the skin, so that the skin is smooth and moist, and the invasion of external bacteria, dust and the like is blocked; the sodium hyaluronate with small molecular weight can permeate into dermis, slightly expand capillary, increase blood circulation, improve metabolism, and promote skin nutrition absorption and waste excretion, thereby preventing skin aging and caring skin. However, sodium hyaluronate is rapidly degraded into small molecular fragments under the action of hyaluronidase in vivo, enters body fluid circulation and is absorbed and metabolized, and the function of locally moisturizing skin is lost; sodium hyaluronate has a half-life of less than 1 day in skin (Laurent et al, ExpPhysiol 1991,76: 695-one 703). Therefore, although the water light injection can quickly supplement sodium hyaluronate on local skin, the water light injection can only supplement water and keep moisture in a short time, and the long-term effects of improving skin quality, delaying aging, recovering young state and the like are difficult to realize.

The cross-linked sodium hyaluronate is a polymer gel obtained by cross-linking and modifying sodium hyaluronate with a cross-linking agent, and can overcome the defects of short retention time of sodium hyaluronate in vivo and the like. Currently, the crosslinking agents used to crosslink sodium hyaluronate mainly include two major classes, namely divinyl sulfone (DVS) and 1, 4-dibutanol diglycidyl ether (BDDE). However, both crosslinking agents have high biological toxicity and potential carcinogenicity, and the crosslinked sodium hyaluronate is a main component of the water light needle and has potential safety risk after being injected into human skin. In addition, the content of the cross-linked sodium hyaluronate gel in the current subcutaneous injection filling type products is very high (generally not less than 20 mg/mL), and the water content is generally not more than 98%. Therefore, the crosslinking modification can delay the degradation and absorption rate of the sodium hyaluronate in vivo, but the water replenishing and moisture retaining performance of the sodium hyaluronate is poor. In addition, the cross-linked sodium hyaluronate gel products also have higher gel strength and can be used after being crushed into particles; meanwhile, due to the larger gel strength and granular appearance of the product, the pushing force of the product is increased, so that the product is difficult to perform shallow surface injection through a water smooth needle extremely fine needle; and the crosslinked sodium hyaluronate gel product has poor fluidity, cannot be uniformly distributed on the shallow surface layer of water light injection, is easy to form bulges and cannot be degraded and eliminated in a short time. Therefore, the current crosslinked sodium hyaluronate gel product has a plurality of limitations for water light injection application.

Disclosure of Invention

Aiming at the existing problems, the invention provides a sodium hyaluronate composite solution composition with synergistic stability and a preparation thereof, wherein the composition comprises non-crosslinked sodium hyaluronate, a hyaluronidase inhibitor and other auxiliary materials, has good stability, realizes the effects of moisturizing in vivo for a long time, and ensures the safety of use. The technical scheme is as follows:

firstly, the invention provides a sodium hyaluronate composite solution composition with synergistic stability effect, which comprises non-crosslinked sodium hyaluronate, a hyaluronidase inhibitor, a solution stabilizer, an antioxidant and a pH regulator; wherein the hyaluronidase inhibitor is an amino acid.

In a preferable technical scheme, in the sodium hyaluronate composite solution composition, the content of the non-crosslinked sodium hyaluronate is 1-10 mg/ml, and the molecular weight of the non-crosslinked sodium hyaluronate is 120-180 ten thousand daltons; the mass ratio of the content of the hyaluronidase inhibitor to the non-crosslinked sodium hyaluronate is 0.0001: 1-1: 1; the mass ratio of the content of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.5: 1-2: 1; the mass ratio of the content of the antioxidant to the non-crosslinked sodium hyaluronate is 0.00025: 1-0.1: 1; the pH regulator regulates the pH value of the hyaluronic acid composite solution composition to be 5.5-7.5.

According to a preferable technical scheme, the hyaluronidase inhibitor is one or a combination of more of arginine, glycine, alanine, L-cysteine hydrochloride and methionine.

Further preferably, the hyaluronidase inhibitor is a combination of arginine and/or L-cysteine in combination with glycine, alanine.

According to the preferable technical scheme, the mass ratio of arginine, L-cysteine and non-crosslinked sodium hyaluronate is 0.05-0.5: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0001-0.5: 1.

Further preferably, the mass ratio of the arginine, the L-cysteine and the non-crosslinked sodium hyaluronate is 0.08-0.4: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0005-0.4: 1.

More preferably, the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.1-0.3: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0006-0.3: 1.

The sodium hyaluronate composite solution composition with synergistic stability is characterized in that the solution stabilizer is one or a combination of any several of sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium metabisulfite, sodium bicarbonate, sodium dihydrogen phosphate and sodium chloride.

Preferably, the solution stabilizer is sodium chloride.

According to a preferable technical scheme, the sodium hyaluronate composite solution composition with synergistic stability has a mass ratio of the content of the solution stabilizer to the non-crosslinked sodium hyaluronate of 0.8-1.5: 1.

Further preferably, the mass ratio of the content of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.9-1.3: 1.

As a preferable technical meansThe antioxidant is nicotinamide and vitamin B ₁₂ One or the combination of any of vitamin C and vitamin E.

Further preferably, the antioxidant is nicotinamide and vitamin B ₁₂ Combinations of (a) and (b).

According to a preferable technical scheme, the mass ratio of the content of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.1: 1, and the vitamin B ₁₂ The mass ratio of the content of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.0025: 1.

Further preferably, the mass ratio of the content of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.05: 1, and the vitamin B ₁₂ The mass ratio of the content of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00125: 1.

More preferably, the mass ratio of the content of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.03: 1, and the vitamin B ₁₂ The mass ratio of the content of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00075: 1.

In a preferable technical scheme, the pH value of the hyaluronic acid composite solution composition is 6.8-7.5.

The sodium hyaluronate composite solution composition with synergistic stability further comprises a diluent, wherein the diluent is selected from one or more of water for injection, 0.9% of sodium chloride, 0.45% of sodium chloride, 2.5% of glucose and 5% of glucose.

The invention further provides a sodium hyaluronate composite solution composition preparation with a synergistic stability effect, which is an injection or a freeze-drying agent, and the components of the sodium hyaluronate composite solution composition comprise the sodium hyaluronate composite solution composition with the synergistic stability effect.

Preferably, the injection is obtained by terminal moist heat sterilization or sterilization filtration, and the endotoxin is less than 0.5 EU/ml; the freeze-drying agent has a water content of 0-1.0%.

The invention has the beneficial effects that:

1) according to the invention, sodium hyaluronate and auxiliary materials for inhibiting hyaluronidase are combined, so that the problems of quick in-vivo hydrolysis and short half-life period of sodium hyaluronate are solved; the retention time of the sodium hyaluronate in the body is prolonged by a non-crosslinking means; compared with the hyaluronidase substances such as adenosine heptapeptide, tea polyphenol, echinacoside and the like, the hyaluronidase inhibitor not only can inhibit the activity of the hyaluronidase, but also has obvious effect; in addition, the problem of pharmacological activity does not exist when the amino acid is adopted to inhibit the hyaluronidase, the method is safe and reliable, the activity of the hyaluronidase can be inhibited, meanwhile, the collagen can be effectively promoted to generate, and the effect of achieving twice the result with half the effort is achieved.

2) The amino acid as the hyaluronidase inhibitor of the present invention is preferably a combination of arginine and/or L-cysteine with glycine and alanine; arginine and L-cysteine are specifically combined with the N tail end of hyaluronidase through the molecular structure characteristics of the arginine and the L-cysteine, so that the effect of inhibiting the activity of the hyaluronidase is achieved; the glycine and the alanine are combined with the macromolecular sodium hyaluronate through a hydrogen bond effect to form steric hindrance of the combination of the hyaluronidase and the sodium hyaluronate, so that the activity of the hyaluronidase for cutting the sodium hyaluronate is inhibited; meanwhile, the pH value of alanine in the selected amino acids is 6.96, the pH value of arginine is 9.67, the pH value of L-cysteine is 5.24, and glycine is 6.21, so that the hyaluronidase can not obtain the optimal pH environment for enzyme digestion (the hyaluronidase can be divided into two according to the optimal pH values thereof, wherein one is acid lysosome hyaluronidase, the optimal pH is 3.0 and is inactive under the neutral condition, and the optimal pH value of the second type of hyaluronidase is neutral and is 7.0), and the effect of inhibiting the enzyme activity can also be achieved to a certain extent.

3) The invention discovers that the non-crosslinked sodium hyaluronate and different amino acids used for inhibiting the sodium hyaluronate are mixed according to a specific proportion, and the inhibition effect of any one amino acid inhibitor on hyaluronidase is superior to that of independently adding any one amino acid inhibitor, so that the mixture of multiple amino acids has the effect of inhibiting 1+1>2 hyaluronidase after the synergistic effect of the two types of amino acids with different action mechanisms. And the mass ratio of the arginine, the L-cysteine and the non-crosslinked sodium hyaluronate is 0.05-0.5: 1; the hyaluronidase inhibition effect is more obvious under the condition that the mass ratio of glycine to alanine to non-crosslinked sodium hyaluronate is 0.0001-0.5: 1.

4) The invention firstly verifies that the sodium hyaluronate macromolecular structure can wrap small molecular substances such as a hyaluronidase inhibitor, a solution stabilizer, an antioxidant, a pH regulator and the like in the composition, so that the physicochemical properties of the small molecules are stable; the coated hyaluronidase inhibitor can protect sodium hyaluronate from being hydrolyzed by hyaluronidase and prolong the retention time of the sodium hyaluronate; the solution stabilizer and the antioxidant increase the stability of the composite solution composition and realize the synergistic stabilization effect.

5) The composition can obtain injection through terminal moist heat sterilization or sterile filtration due to outstanding excellent performance in stability, and can also obtain freeze-dried preparation through freeze drying, the endotoxin of the obtained injection is less than 0.5EU/ml, the moisture of the finished freeze-dried preparation is only 0-1.0%, and the safety of the composition is further improved. The obtained injection and freeze-drying agent are stored at room temperature, the headspace oxygen is lower than 1.0 percent and the dissolved oxygen is less than 1ppm after the injection and freeze-drying agent are placed in a penicillin bottle, and the injection and freeze-drying agent has good industrial production value and economic value.

Drawings

FIG. 1 is a chemical structural formula of sodium hyaluronate in the sodium hyaluronate composite solution composition with synergistic stability effect of the present invention;

FIG. 2 is a graph showing the results of the plate-type enzyme activity assay of the complex solution composition of the present invention containing different amino acids;

FIG. 3 is a standard curve of absorbance-enzyme content of hyaluronidase assay for complex solution compositions containing different amino acids according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments.

Example 1

The embodiment is a sodium hyaluronate composite solution composition with synergistic stability, which comprises non-crosslinked sodium hyaluronate, a hyaluronidase inhibitor, a solution stabilizer, an antioxidant and a pH regulator; wherein the hyaluronidase inhibitor is an amino acid. In the research process, the sodium hyaluronate is rapidly degraded into small molecular fragments under the action of hyaluronidase in vivo, the small molecular fragments enter body fluid circulation and are absorbed and metabolized, and the function of locally moisturizing skin is lost. Therefore, it is important to find safe auxiliary materials capable of inhibiting the activity of hyaluronidase. Experiments show that amino acid, adenosine heptapeptide, tea polyphenol and echinacoside can inhibit the activity of hyaluronidase activity, but polyglutamic acid, adenosine heptapeptide, tea polyphenol and echinacoside have far lower effect of inhibiting the activity of enzyme than amino acid, and have certain pharmacological activity, and from the perspective of safety, amino acid is the better choice.

In the embodiment, amino acid is used as a hyaluronidase inhibitor and is used as an auxiliary material to be combined with non-crosslinked sodium hyaluronate, so that the problems of fast in-vivo hydrolysis and short half-life period of sodium hyaluronate are solved; the purpose of prolonging the retention time of the non-crosslinked sodium hyaluronate in the body is achieved, and the adopted amino acid is used as the hyaluronidase inhibitor, so that the hyaluronidase inhibitor not only has good activity of inhibiting the activity of hyaluronidase and is safe and reliable, but also can effectively promote the generation of collagen while inhibiting the activity of the hyaluronidase, and has the effect of achieving twice the result with half the effort. In addition, in the embodiment, the sodium hyaluronate macromolecular structure can wrap small molecular substances such as a hyaluronidase inhibitor, a solution stabilizer, an antioxidant and a pH regulator in the composition, so that the small molecules have stable physicochemical properties; the coated hyaluronidase inhibitor can protect sodium hyaluronate from being hydrolyzed by hyaluronidase and prolong the retention time of the sodium hyaluronate; the solution stabilizer and the antioxidant increase the stability of the composite solution composition and realize the synergistic stabilization effect.

In the embodiment, the content of the non-crosslinked sodium hyaluronate is 1-10 mg/ml, and the molecular weight of the non-crosslinked sodium hyaluronate is 120-180 ten thousand daltons; the mass ratio of the content of the hyaluronidase inhibitor to the non-crosslinked sodium hyaluronate is 0.0001: 1-1: 1; the mass ratio of the content of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.5: 1-2: 1; the mass ratio of the content of the antioxidant to the non-crosslinked sodium hyaluronate is 0.00025: 1-0.1: 1; the pH regulator regulates the pH of the hyaluronic acid composite solution composition to be 5.5-7.5, preferably 6.8-7.5.

In this embodiment, the hyaluronidase inhibitor is one or a combination of arginine, glycine, alanine, L-cysteine salt, or methionine. Preferably arginine and or L-cysteine in combination with glycine, alanine. Wherein arginine and L-cysteine are specifically combined with the N terminal of hyaluronidase through the molecular structure characteristics of the arginine and the L-cysteine, so that the effect of inhibiting the activity of the hyaluronidase is achieved; the glycine and the alanine are combined with macromolecular sodium hyaluronate through a hydrogen bond effect to form steric hindrance caused by the combination of the hyaluronidase and the sodium hyaluronate, so that the activity of the hyaluronidase for cutting the sodium hyaluronate is inhibited; meanwhile, the pH value of alanine in the selected amino acids is 6.96, the pH value of arginine is 9.67, the pH value of L-cysteine is 5.24, and glycine is 6.21, so that the hyaluronidase can not obtain the optimal pH environment for enzyme digestion (the hyaluronidase can be divided into two according to the optimal pH values thereof, wherein one is acid lysosome hyaluronidase, the optimal pH is 3.0 and is inactive under the neutral condition, and the optimal pH value of the second type of hyaluronidase is neutral and is 7.0), and the effect of inhibiting the enzyme activity can also be achieved to a certain extent. And glycine and alanine are two types of amino acids with the highest content in the collagen, arginine is the second amino acid in the collagen to glycine, alanine and glutamic acid, arginine is essential amino acid for nutrition, a human body can only synthesize part, and food is needed to be provided when the long-term intake is insufficient or the demand is increased. Therefore, the glycine, the alanine and the arginine are added into the sodium hyaluronate composite solution for injection, so that the activity of hyaluronidase can be inhibited, and the generation of collagen can be effectively promoted.

In a preferred embodiment, the mass ratio of arginine, L-cysteine and non-crosslinked sodium hyaluronate is 0.05-0.5: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0001-0.5: 1; more preferably, the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.08-0.4: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0005-0.4: 1. More preferably, the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.1-0.3: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0006-0.3: 1.

In this embodiment, the solution stabilizer is one or a combination of any several of sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium metabisulfite, sodium bicarbonate, sodium dihydrogen phosphate and sodium chloride; sodium chloride is preferred. The mass ratio of the content of the solution stabilizer to the non-crosslinked sodium hyaluronate is preferably 0.8-1.5: 1; more preferably 0.9 to 1.3: 1.

In this embodiment, the antioxidant is nicotinamide and vitamin B ₁₂ One or the combination of any more of vitamin C or vitamin E, preferably nicotinamide and vitamin B ₁₂ Combinations of (a) and (b). The mass ratio of the content of nicotinamide to the non-crosslinked sodium hyaluronate is preferably 0.01-0.1: 1, and the vitamin B ₁₂ The mass ratio of the content of (A) to the non-crosslinked sodium hyaluronate is preferably 0.00025-0.0025: 1. Further preferably, the mass ratio of the content of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.05: 1, and the vitamin B ₁₂ The mass ratio of the content of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00125: 1; more preferably, the mass ratio of the content of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.03: 1, and the vitamin B ₁₂ The mass ratio of the content of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00075: 1.

In this embodiment, the pH adjuster is one or any two or a combination of two or more selected from hydrochloric acid, sulfuric acid, acetic acid, methanesulfonic acid, malic acid, maleic acid, citric acid and salts thereof, phosphoric acid and salts thereof, lactic acid and salts thereof, tartaric acid and salts thereof, and succinic acid and salts thereof, and hydrochloric acid is preferred. In addition, the sodium hyaluronate compound solution composition of the present embodiment may further include a diluent, wherein the diluent is selected from one or a combination of several of water for injection, 0.9% sodium chloride, 0.45% sodium chloride, 2.5% glucose, and 5% glucose. The sodium hyaluronate composite solution composition with synergistic stability effect can be prepared into injection or freeze-dried preparation, wherein the injection is obtained by terminal moist heat sterilization or sterilization filtration, and the endotoxin is less than 0.5 EU/ml; the freeze-drying agent contains 0-1.0% of water.

In this embodiment, the reconstitution time of the lyophilized preparation after reconstitution with water for injection or physiological saline is less than 45 seconds; the solution after the freeze-drying agent is reconstructed and the prepared injection can keep stability (API degradation degree is not higher than 0.5 wt%) for at least 9 hours under high temperature (45-60 ℃) and natural illumination; and the injection and the freeze-drying agent can be stored at room temperature, the content of related substances is not more than 1.0 percent after the injection and the freeze-drying agent are subjected to accelerated lofting for 6 months (40 +/-2 ℃/75 +/-5 percent RH), the headspace oxygen is lower than 1.0 percent after the injection and the freeze-drying agent are placed in a penicillin bottle, and the dissolved oxygen is less than 1 ppm.

Example 2

This example is to examine the inhibition effect of different hyaluronidase inhibitors (i.e. different amino acids and concentrations) added to the sodium hyaluronate composite solution composition described in the examples on the hyaluronidase activity and the effect on the stability of the composition solution

The experimental formula is shown in formula 1-6 in table 1.

TABLE 1 Hyaluronidase inhibitor formulations at different amino acids and concentrations

The preparation process comprises the following steps: according to the formula, weighing the components, adding part of water, heating to 60 ℃, stirring and dissolving, adjusting the pH to 6.5 by hydrochloric acid, complementing the water for injection to 10000mg, filtering by 0.22 unit of PVDF filter membrane, subpackaging and sterilizing by damp heat; two portions of the composite solution composition were prepared for each formulation and were ready for use.

Hyaluronidase inhibition assay: bovine testicular hyaluronidase is dissolved in normal saline to make a solution containing 0.1mg per 1ml, and added to one part of the composite solution composition of the above formula, and the other part is used as a control without enzyme. Then, the experimental group and the control group were placed together on a shaker at 37 ℃ for 12 hours, and then the phenomenon of the sample was observed and the viscosity of the sample was measured. The results are shown in Table 2.

Table 2 shows the hyaluronidase inhibition experiment results of formulas 1 to 6

The experimental results show that:

firstly, in the sample added with the glycine and the alanine independently, after the hyaluronidase is added, the property and the viscosity of the sample are changed greatly compared with the control sample without the enzyme, and the viscosity of the sample is changed slightly compared with the formula sample only added with the enzyme, which indicates that the activity of the glycine or the alanine for inhibiting the hyaluronidase independently has no obvious arginine and L-cysteine effect.

And secondly, in the sample with both glycine and alanine, after hyaluronidase is added, the properties and viscosity of the sample are almost unchanged compared with the sample without enzyme, which shows that the mixing ratio of glycine and alanine can better inhibit the activity of hyaluronidase after being added alone.

And thirdly, the sample with the L-cysteine and the L-arginine added independently has almost no change in properties and viscosity compared with the sample without the enzyme, which shows that the L-cysteine and the L-arginine can prepare the activity of the hyaluronidase.

Example 3

This example includes three experiments to further investigate the inhibitory effect of different amino acid combinations on hyaluronidase activity. The experimental formulation is shown in table 3.

TABLE 3 different amino acid formulations

The preparation process comprises the following steps: mixing the components for preparation.

This example uses two methods to test the inhibitory effect of different combinations of amino acids on hyaluronidase activity.

Experiment 1: the enzyme activity detection process by plate diffusion: preparing an agarose plate containing high-molecular hyaluronic acid; punching a plurality of sample adding holes with the diameter of 6mm on the agarose plate by using a sterile puncher; and adding 7-10 formulations, blank enzyme solution and water for injection into the sample adding holes respectively in an equal amount, wherein the sample adding amount is 50 mu L. After sample application, the plate was placed in an incubator at 37 ℃ for about 20 hours, covered with 10ml of a 10% (w/v) cetylpyridinium chloride aqueous solution, and developed after about 10 minutes, as a result of the experiment: see fig. 1.

The results show that: the sample of the blank enzyme solution has obvious transparent circles on the flat plate, the transparent circles of the blank solution with different enzyme concentrations are increased according to the increase of the enzyme, and other formula samples do not appear, which indicates that the amino acid can inhibit the activity of the hyaluronidase.

Experiment 2: the observation of the inhibitory effect of amino acids on hyaluronidase activity was performed by using the 1207 hyaluronidase assay of the fourth pharmacopoeia edition 2020:

preparing a standard curve, namely taking 12 test tubes with the same size, and adding 2 test tubes of standard solution of 0.00ml, 0.10ml, 0.20ml, 0.30ml, 0.40ml and 0.50ml in sequence; sequentially and correspondingly adding 0.50ml, 0.40ml, 0.30ml, 0.20ml, 0.10ml and 0.00ml of hydrolyzed gelatin diluent, sequentially adding 0.50ml of potassium hyaluronate solution every 30 seconds to ensure that the total volume of each tube is 1.00ml, shaking up, and placing in a water bath at 37 +/-0.5 ℃; after each tube is accurately kept warm for 30 minutes, the tubes are taken out at intervals of 30 seconds, 4.0ml of serum solution is immediately and sequentially added, the tubes are shaken up, the tubes are placed at room temperature for 30 minutes, the tubes are shaken up, and the absorbance is measured at the wavelength of 640 nm; meanwhile, 0.50ml of phosphate buffer solution is used to replace the potassium hyaluronate solution, 0.50ml of hydrolyzed gelatin diluent is added, the mixture is shaken up, and the operation is carried out according to the method from ' placing in a water bath at 37 +/-0.5 ℃ to ' using as a blank '. And (3) drawing a standard curve by taking the absorbance as an ordinate and the titer (unit) of the standard solution as an abscissa.

The determination method comprises sequentially adding test solution 0.20ml, 0.30ml and 0.40ml into 6 test tubes with the same size, 2 test tubes respectively; and sequentially and correspondingly adding 0.30ml, 0.20ml and 0.10ml of hydrolyzed gelatin diluent, and measuring according to the method from the point that 0.50ml of potassium hyaluronate solution is added every 30 seconds under the preparation item of a standard curve, dividing the measured result by the weight (mg) of the test sample after the titer (unit) is checked on the standard curve, and calculating the average number of 6 parts of the test sample, namely the titer (unit) of the hyaluronidase.

The standard curves and experimental results are shown in fig. 2 and table 4.

TABLE 4 absorbance values of different formulations and corresponding enzymatic potency values

The results show that: the addition of amino acid reduces the original potency of the enzyme from 120 NFU/mg to less than ten percent, and the formula 8 has the most obvious effect of inhibiting the activity of the hyaluronidase by mixing L-arginine, L-cysteine, glycine and alanine; the effect of inhibiting the enzymatic hydrolysis of the hyaluronic acid is also more remarkable when the L-arginine and the L-cysteine in the formula 9 and the formula 10 are respectively and independently mixed with the glycine and the alanine, which shows that the hyaluronic acid enzymatic activity can be better inhibited when the amino acid for inhibiting the enzymatic activity of the hyaluronic acid is mixed and added according to different mechanisms.

Experiment 3: the inhibition effect of different hyaluronidase inhibitors on the hyaluronidase activity is examined, and the experimental formula is shown in table 5.

TABLE 5 different Hyaluronidase inhibitor formulations

The experiment and measurement method of experiment 3 are the same as those mentioned in experiment 2, and the standard curve and the experimental results are shown in FIG. 2 and Table 6.

TABLE 6 absorbance values and corresponding enzyme potency values for different hyaluronidase inhibitor formulations

The results show that: amino acids, polyglutamic acid, adenosine heptapeptide, tea polyphenol and echinacoside can inhibit the activity of the hyaluronidase activity, but the polyglutamic acid, the adenosine heptapeptide, the tea polyphenol and the echinacoside have far lower effect of inhibiting the activity of the enzyme than the amino acids, and have certain pharmacological activity, and the amino acids are preferred from the safety point of view.

Through the three experiments, the amino acid has a strong effect of inhibiting the activity of the hyaluronidase, can delay the degradation speed of the sodium hyaluronate by the enzyme, and can maintain the physiological effect of the sodium hyaluronate to the maximum extent.

Example 4

This example is two experiments, the purpose of which is to examine the effect of stabilizers on the stability of formulations.

1) The effect of different stabilizers on the stability of the formulations was examined and the experimental formulations are shown in table 7.

TABLE 7 different stabilizer formulations

The preparation process comprises the following steps: the preparation method of the same example 2 is that the components are weighed, water is added into the components, the mixture is heated to 60 ℃, stirred and dissolved, hydrochloric acid is adjusted to 7.0, water for injection is complemented to 10000mg, 0.22un PVDF filter membrane is used for filtration, and split charging is carried out after moist heat sterilization.

And (3) placing the prepared composite solution at 60 ℃ for 0, 5 and 10 days to detect the content of the sodium hyaluronate and related substances. The results are shown in Table 8.

TABLE 8 results of the effect of different stabilizers on the stability of the formulations

The experimental results are as follows: compared with a plurality of stabilizers, the effect of sodium chloride on the stability of the composite solution composition is more obvious than that of other stabilizers after the stabilizers with the same quality are added, the stability of the solution is further enhanced, and the sodium chloride is the main component of normal saline and has the advantage that other stabilizers cannot be replaced in the aspect of safety.

2) The effect of different amounts of sodium chloride on the stability of the formulation was examined. The experimental formulation is shown in table 7.

TABLE 9 formulation of different amounts of the same stabilizer

The preparation process comprises the following steps: the preparation method of the same example 2 is that the components are weighed, water is added into the components, the mixture is heated to 60 ℃, stirred and dissolved, hydrochloric acid is adjusted to 7.0, water for injection is made up to 10000mg, 0.22un PVDF filter membrane is used for filtration, and split charging is carried out after moist heat sterilization.

TABLE 10 results of the effect of different stabilizers on the stability of the formulations

The experimental results are as follows: firstly, compared with the formula 20 without the stabilizer, compared with the formula 6, the formula content with the stabilizer and the variation range of related substances are smaller. Different qualities of sodium chloride were added to the formulation and it was found that the stability of the composite solution composition was further enhanced with increasing sodium chloride.

Secondly, sodium hyaluronate is not added in the formula 21, only small molecular substances and stabilizing agents are added, and influence factor experiments can find that related substances are in an ascending trend, which shows that the stability of small molecules is reduced to a certain extent if no macromolecular sodium hyaluronate is used for coating a screen. This example demonstrates the synergistic stabilizing effect of a stabilizer and a macromolecular sodium hyaluronate for a solution composition.

Example 5

This example is to examine the effect of various antioxidants added on headspace oxygen in the formulation bottle, dissolved oxygen content in the solution and related substances. The experimental formulation is shown in table 11.

TABLE 11 different antioxidant formulations

The preparation process comprises the following steps: before preparation, filling nitrogen into water for injection, filling nitrogen during preparation, weighing each component, adding part of water for injection, heating to 60 ℃, stirring and dissolving, adjusting hydrochloric acid to 7.0, complementing the water for injection to 10000mg, controlling the content of dissolved oxygen in the solution to be lower than 1ppm, filtering by 0.22un PVDF filter membrane, filling by filling nitrogen, performing moist heat sterilization, and controlling the content of headspace oxygen in a bottle to be lower than 1.0%. The intermediate solution is placed at 60 ℃ for influencing factor experiments, and headspace oxygen in a formula bottle, the content of dissolved oxygen in the solution and related substances are detected in 0 to 10 days. The results are shown in Table 12.

TABLE 12 influence factor test results for different antioxidant formulations

The experimental results show that: the headspace oxygen and dissolved oxygen contents of the solution composition of the formula 29 without the antioxidant are obviously reduced in the influence factor lofting process, but the growth of related substances is obvious, the effective components and the auxiliary components of the composition are supposed to react with oxygen to generate a certain amount of related substances, the headspace oxygen and dissolved oxygen contents of the composition with the antioxidant are not obviously reduced in the influence factor lofting process, and meanwhile, the related substances grow slowly, which shows that the antioxidant inhibits the oxidation reaction of the solution composition, and the stability of the whole solution system is promoted.

Example 6

This example is to examine the irritation of the sodium hyaluronate composite solution composition proposed in example 1 to the skin of animals.

In this example, a skin irritation test study was conducted using rabbits as test subjects to examine the potential of the composition solution to produce an irritation response under the test conditions.

Experimental samples: this example used the sodium hyaluronate sample prepared from formulation 22 of example 5.

Experimental animals: healthy new zealand white rabbits 3.

And (3) experimental operation: a sample of the composition solution was placed in direct contact with the skin on both sides of the spine of the rabbit for a single 24h, and the gauze piece was mounted. Erythema and edema were scored at the contact sites at (24. + -.2) h, (48. + -.2) h, and (72. + -.2) h after contact, and the primary stimulation index (PII) was calculated.

Evaluation indexes: the scoring criteria were as per table 13.

TABLE 13 skin response score criteria

The test results are shown in Table 14.

TABLE 14 skin response score results

From the above experimental results, it can be seen that under the present experimental conditions, the skin reaction stimulation index of the sodium hyaluronate composite solution composition for rabbits was 0. The composite solution combination skin test is safe and qualified.

Example 7

This example is to examine the degradation of the sodium hyaluronate composite solution composition proposed in example 1 under the skin of animals.

Purpose of the experiment: a mouse is taken as a test object to carry out subcutaneous sodium hyaluronate degradation test research, and the subcutaneous degradation conditions of the composition solution and the sodium hyaluronate aqueous solution are compared.

Experimental samples: a sodium hyaluronate composition solution sample was prepared according to formulation 22 in example 5, while a sodium hyaluronate solution control sample prepared according to formulation 6 in example 2; and the hyaluronic acid content of the subcutaneous tissue of the mouse is measured by using physiological saline as a blank control.

Experimental animals: healthy mice were 36.

The experimental method comprises the following steps: 36 mice were randomly divided into 3 groups of 12 mice each, one group injected with a solution sample of composition formulation 22, a second group injected with a sample of control formulation 6, and a third group injected with an equal amount of saline. Each group of mice was divided into a 2-week test group, a 3-week test group and a 4-week test group. After each group of mice is injected subcutaneously, the experimental mice are killed at the experimental detection points, skin tissues at the injection part of 1cm x 1cm are taken, pancreatin solution is added for digestion, the supernatant is taken after centrifugation, the content of the sodium hyaluronate in the supernatant is detected by referring to a sodium hyaluronate detection method in YYT 0308-2015 medical sodium hyaluronate gel, the subcutaneous hyaluronic acid content of the mice is deducted, and the subcutaneous sodium hyaluronate residual content of the mice is calculated.

The calculation method comprises the following steps: mouse subcutaneous sodium hyaluronate residual content = (content of sodium hyaluronate in supernatant-mouse subcutaneous hyaluronic acid content per se)/amount of hyaluronic acid in injection sample × 100%.

The results are shown in Table 15.

TABLE 15 residual sodium hyaluronate content results

From the above experimental results, it can be known that under the experimental conditions, the sodium hyaluronate composite solution composition has a great difference from the sodium hyaluronate solution in the content of degradation residues under the skin of mice. The addition of the amino acid can effectively delay the degradation time of the sodium hyaluronate in the body.

According to the invention, sodium hyaluronate and auxiliary materials for inhibiting hyaluronidase are combined for the first time, so that the problems of quick in-vivo hydrolysis and short half-life period of sodium hyaluronate are solved; the hyaluronidase inhibitor is amino acid, has good performance of inhibiting the activity of hyaluronidase, is safe and reliable, can effectively promote the generation of collagen while inhibiting the activity of hyaluronidase, and has the effect of achieving twice the result with half the effort. Preferred hyaluronidase inhibitors of the present invention are combinations of arginine and/or L-cysteine with glycine and alanine; wherein arginine and L-cysteine are specifically combined with hyaluronidase through the molecular structure characteristics of the arginine and the L-cysteine, so that the effect of inhibiting the activity of the hyaluronidase is achieved; the glycine and the alanine are combined with the macromolecular sodium hyaluronate through a hydrogen bond effect to form steric hindrance of the combination of the hyaluronidase and the sodium hyaluronate, so that the activity of the hyaluronidase for cutting the sodium hyaluronate is inhibited; meanwhile, the hyaluronidase cannot obtain the optimal pH environment for enzyme digestion due to the special acidity and alkalinity of amino acid, and the effect of inhibiting the enzyme activity is achieved to a certain extent.

Meanwhile, the invention verifies that the sodium hyaluronate macromolecular structure can wrap small molecular substances such as a hyaluronidase inhibitor, a solution stabilizer, an antioxidant, a pH regulator and the like in the composition for the first time, so that the activity of the small molecules is stable; the coated hyaluronidase inhibitor can protect sodium hyaluronate from being hydrolyzed by hyaluronidase and prolong the retention time of the sodium hyaluronate; the solution stabilizer and the antioxidant increase the stability of the composite solution composition, realize the synergistic and stable effect and have good industrial production value and economic value.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Furthermore, it should be understood that although the present specification describes embodiments, this does not include only one embodiment, and such description is for clarity only, and those skilled in the art should be able to make the specification as a whole, and the embodiments may be appropriately combined to form other embodiments understood by those skilled in the art.

Claims

1. A synergistically stabilized sodium hyaluronate composite solution composition characterized by: the sodium hyaluronate composite solution composition consists of non-crosslinked sodium hyaluronate, a hyaluronidase inhibitor, a solution stabilizer, an antioxidant and a pH regulator;

wherein the content of the first and second substances,

the content of the non-crosslinked sodium hyaluronate is 1-10 mg/ml, and the molecular weight of the non-crosslinked sodium hyaluronate is 120-180 ten thousand daltons;

the hyaluronidase inhibitor is a combination of arginine and/or L-cysteine with glycine, alanine;

the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.05-0.5: 1;

the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0001-0.5: 1;

the mass ratio of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.5: 1-2: 1;

the mass ratio of the antioxidant to the non-crosslinked sodium hyaluronate is 0.00025: 1-0.1: 1;

the pH regulator regulates the pH value of the sodium hyaluronate composite solution composition to be 5.5-7.5.

2. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 1 wherein: the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.08-0.4: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0005-0.4: 1.

3. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 2 characterized in that: the mass ratio of the arginine to the L-cysteine to the non-crosslinked sodium hyaluronate is 0.1-0.3: 1; the mass ratio of the glycine to the alanine to the non-crosslinked sodium hyaluronate is 0.0006-0.3: 1.

4. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 1 wherein: the solution stabilizer is one or the combination of any more of sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium metabisulfite, sodium bicarbonate, sodium dihydrogen phosphate or sodium chloride.

5. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 4 wherein: the solution stabilizer is sodium chloride.

6. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 5 wherein: the mass ratio of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.8-1.5: 1.

7. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 6 wherein: the mass ratio of the solution stabilizer to the non-crosslinked sodium hyaluronate is 0.9-1.3: 1.

8. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 1, wherein: the antioxidant is nicotinamide and vitamin B ₁₂ One or the combination of any of vitamin C and vitamin E.

9. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 8 wherein: the antioxidant is nicotinamide and vitamin B ₁₂ Combinations of (a) and (b).

10. The synergistically stabilized sodium hyaluronate associate solution of claim 9A liquid composition characterized by: the mass ratio of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.1: 1, and the vitamin B ₁₂ The mass ratio of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.0025: 1.

11. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 10 wherein: the mass ratio of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.05: 1, and the vitamin B ₁₂ The mass ratio of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00125: 1.

12. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 11 wherein: the mass ratio of the nicotinamide to the non-crosslinked sodium hyaluronate is 0.01-0.03: 1, and the vitamin B ₁₂ The mass ratio of the sodium hyaluronate to the non-crosslinked sodium hyaluronate is 0.00025-0.00075: 1.

13. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 1 wherein: the pH value of the sodium hyaluronate composite solution composition is 6.8-7.5.

14. The synergistically stabilized sodium hyaluronate composite solution composition according to claim 1 wherein: the sodium hyaluronate composite solution composition is also added with a diluent, wherein the diluent is selected from one or a combination of more of water for injection, 0.9% of sodium chloride, 0.45% of sodium chloride, 2.5% of glucose and 5% of glucose.

15. A synergistically stabilized sodium hyaluronate composite solution composition formulation characterized by: the preparation is an injection or a freeze-dried preparation, and the components of the preparation comprise the sodium hyaluronate composite solution composition with synergistic stability of any one of claims 1 to 14.

16. The synergistically stabilized sodium hyaluronate composite solution composition formulation according to claim 15, characterized in that: the injection is obtained by terminal moist heat sterilization or sterilization filtration, and the endotoxin is less than 0.5 EU/ml; the freeze-drying agent contains 0-1.0% of water.